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The Journal of Neuroscience, December 15, 2000, 20(24):8965-8971
Induction of Cyclin-Dependent Kinase 5 in the Hippocampus by
Chronic Electroconvulsive Seizures: Role of  FosB
Jingshan
Chen,
Yajun
Zhang,
Max B.
Kelz,
Cathy
Steffen,
Eugenius S.
Ang,
Ling
Zeng, and
Eric J.
Nestler
Laboratory of Molecular Psychiatry, Yale University School of
Medicine and Connecticut Mental Health Center, New Haven, Connecticut
06508
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ABSTRACT |
The transcription factor  FosB is induced in the hippocampus and
other brain regions by repeated electroconvulsive seizures (ECS), an
effective antidepressant treatment. The unusually high stability of
this protein makes it an attractive candidate to mediate some of the
long-lasting changes in the brain caused by ECS treatment. To
understand how FosB might alter brain function, we examined the gene
expression profiles in the hippocampus of inducible transgenic mice
that express FosB in this brain region by the use of cDNA expression
arrays that contain 588 genes. Of the 430 genes detected, 20 genes were
consistently upregulated, and 14 genes were downregulated, by >50%.
One of the upregulated genes is cyclin-dependent kinase 5 (cdk5). On
the basis of its purported role in regulating neuronal structure, we
studied directly whether cdk5 is a true target for FosB.
Upregulation of cdk5 immunoreactivity in the hippocampus was confirmed
by Western blotting in the FosB-expressing transgenic mice as well
as in rats treated chronically with ECS. Chronic ECS treatment also
increased, in the hippocampus, the phosphorylation state of tau, a
microtubule-associated protein that is a known substrate for cdk5. A
1.6 kb fragment of the cdk5 promoter was cloned, and activity of the
promoter was found to be increased after overexpression of FosB in
cell culture. Moreover, mutation of the single consensus activator protein-1 site contained within the cdk5 promoter fragment completely abolished activation of the promoter by FosB. Together, these results suggest that cdk5 is one target by which FosB produces some
of its physiological effects in the hippocampus and thereby mediates
certain long-term consequences of chronic ECS treatment.
Key words:
cdk5; FosB; hippocampus; electroconvulsive seizures; transcription factors; antidepressant treatments; inducible transgenic
mice; gene expression
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INTRODUCTION |
Repeated administration of
electroconvulsive seizures (ECS) is one of the most effective
treatments for depression; yet the mechanisms by which it exerts its
clinical effects remain incompletely understood. Regulation of gene
expression by specific transcription factors may be one important
mechanism involved, because the beneficial effects of ECS treatment can
last long after the last ECS. Two transcription factors that are
induced by chronic ECS in the hippocampus, a brain region implicated in
depression and antidepressant treatments (Duman et al., 1999 ), are cAMP
response element binding protein (Nibuya et al., 1996) and
FosB (Hope et al., 1994a; Chen et al., 1995, 1997). FosB is of
particular interest because of its unique temporal properties (Nestler
et al., 1999). FosB is induced in the hippocampus and in certain
regions of cerebral cortex only after repeated ECS administration.
Moreover, after being induced, it persists in the brain for relatively
long periods of time (several weeks) because of its extraordinary
stability. Thus, FosB could be an important mediator of some of the
long-lasting adaptive changes that chronic ECS treatment produces in
the hippocampus.
FosB is a truncated splice variant of the fosB
gene. FosB heterodimerizes with JunD and, to a lesser extent,
JunB to form activator protein-1 (AP-1) complexes that bind to specific
AP-1 sites contained within the 5'-promoter regions of certain genes (Chen et al., 1995). FosB-containing AP-1 complexes are reported to
act as both transcriptional repressors and activators in
vitro, depending on the gene and cell type involved (Dobrazanski
et al., 1991; Nakabeppu and Nathans, 1991; Yen et al., 1991; Chen et
al., 1997). Identification of specific target genes via which FosB produces its physiological effects in vivo is an important
step in understanding the functional role played by this novel
transcription factor. One approach to answer this question is to
evaluate genes with known AP-1 sites as potential physiological targets
for FosB. This approach has succeeded recently in identifying two
glutamate receptor subunits as possible targets for FosB: the
NR1 subunit of NMDA receptors (Hiroi et al., 1998) and the GluR2
subunit of AMPA receptors (Kelz et al., 1999).
A more open-ended and complementary approach is to use differential
gene expression analysis to identify genes that are regulated by
FosB in vivo. To do this effectively, however, it must be possible to induce FosB selectively within brain regions of interest in adult animals. We have accomplished this goal by use of the tetracycline-regulated gene expression system and have constructed bitransgenic mice that express FosB in selected brain regions, including the hippocampus, in an inducible and brain region-specific manner (Chen et al., 1998; Kelz et al., 1999). Such inducible transgenic
mice represent ideal tools with which to search for physiological
targets for FosB, because they avoid the developmental compensations
that complicate many conventional transgenic and knock-out animals.
In the present study, we used cDNA arrays to analyze gene expression
patterns in the hippocampus of these FosB-expressing mice as a means
of identifying novel targets for this transcription factor in
vivo. One of the many genes found to be consistently regulated in
the hippocampus after FosB expression was that encoding cyclin-dependent kinase 5 (cdk5). We further pursued regulation of
cdk5, because this protein appeared to be of particular interest as a
putative target for FosB in the hippocampus (see Discussion). In
agreement with the DNA array finding, we show here that cdk5 does
indeed appear to be a physiological target of FosB, and for
chronic ECS treatment, in the hippocampus. The results thus illustrate
the usefulness of combining an open-ended DNA array-based approach with
inducible, tissue-specific transgenic mice to identify novel targets
for FosB in the brain.
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MATERIALS AND METHODS |
FosB-expressing mice. Bitransgenic 11A or 1A mice,
which express FosB in an inducible and brain region-specific manner
(including the hippocampus), were used in these studies (Chen et al.,
1998; Kelz et al., 1999). In initial experiments, 1A mice that
contained both transgenes (NSE-tTA and TetOp-FosB; see Fig. 1) were
compared with littermates that contained only one transgene (NSE-tTA). In later experiments, 11A mice were used. The mice were conceived and
raised on doxycycline (100 µg/ml) in the drinking water, which is
known to suppress FosB expression completely (Chen et al., 1998).
Half of the littermates were removed from doxycycline at 3-4 weeks of
age and were used 6 weeks later, at which time FosB expression is
known to be turned on. The hippocampus was removed from decapitated
mice by gross dissection. 1A mice were used in initial studies, because
this line expresses much higher levels of FosB compared with the 11A
mice, although the pattern of expression between the two lines appears
to be equivalent (Chen et al., 1998; Kelz et al., 1999).
cDNA expression arrays. Total RNA was isolated from the
hippocampus of bitransgenic mice, one group maintained on doxycycline and the other group removed from doxycycline, by the use of an RNAqueous phenol-free total RNA isolation kit (Ambion, Austin, TX).
Poly(A+) RNA was isolated from the total
RNA by the use of the Oligotex mRNA isolation kit (Qiagen, Hilden,
Germany). The poly(A+) RNA was used
as a template for the synthesis of
32P-labeled cDNA probes. The cDNA probes
(1500 cpm/µl) were hybridized to Atlas mouse cDNA
expression arrays (Clontech, Cambridge, UK). The arrays were then
exposed to a phosphorimaging screen for 16-24 hr, and the
hybridization signal was analyzed with a Bio-Rad (Hercules, CA) GS-363 PhosphorImager.
ECS treatments. Male Sprague Dawley rats (initial weight,
~200 gm; Charles River Laboratories, Wilmington, MA) were used for all experiments. ECS was administered, as before (Hope et al., 1994a),
via ear clip electrodes (45 mA; 0.3 sec). Chronically treated animals
received a single ECS daily for 10 d and were used 18 hr later.
Control animals received chronic sham treatment, in which electrodes
were clipped onto the rats' ears but no current was applied. The
hippocampus was obtained by gross dissection.
Gel shift assays. Gel shift assays were performed on the
basis of published procedures (Hope et al., 1994a). Mouse or rat hippocampus was homogenized with Dounce homogenizers in 20 vol of
electrophoretic mobility shift assay (EMSA) buffer: 20 mM
HEPES, pH 7.9, 0.4 M NaCl, 20% glycerol, 5 mM
MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 10 µg/ml leupeptin, 0.1 mM p-aminobenzamidine, 1 µg/ml pepstatin, 0.5 mM PMSF, and 5 mM DTT. The
homogenates were incubated on ice for 30 min before centrifugation at
15,000 × g for 20 min at 4°C. Aliquots of
supernatants (containing 20 µg of protein) were incubated at 20°C
for 20 min with 1 µg of poly(dI-dC)·poly(dI-dC), 40 µg of
bovine serum albumin, 10 mM Tris-HCl, pH 7.9, 10 mM KCl, 1 mM EDTA, 4%
glycerol, and 1 ng of the radioactively labeled AP-1 probe. The samples
were incubated for 20 min at 20°C and electrophoresed at 150 V for 2 hr in a nondenaturing 6% acrylamide and 0.16%
N,N'-methylenebisacrylamide gel containing 25 mM Tris-borate buffer, pH 8.3, 1 mM EDTA, and 1.6% glycerol. The gels were dried
and exposed to x-ray film. Levels of AP-1 binding were quantified by
measuring the optical density of specific bands by the use of an image
analysis system with NIH Image software, version 1.41.
Western blotting. Western blotting was performed as
described previously (Hope et al., 1994b). Mouse or rat hippocampus was homogenized in Dounce homogenizers in 10 vol of EMSA buffer. Aliquots of these cellular extracts (containing 50 µg of protein) were then
applied to a 10% acrylamide and 0.27%
N,N'-methylenebisacrylamide resolving gel for
SDS-PAGE overnight at 75 V and electrotransferred to
nitrocellulose filters at 200 mA for 3 hr. The blots were incubated in
blocking buffer, with four changes of 15 min each, containing 2%
nonfat dry milk powder in PBS-Tween (10 mM sodium
phosphate, pH 7.4, 150 mM NaCl, and 0.1% Tween
20) and incubated overnight at 4°C in a 1:200,000 dilution of
anti-cdk5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA),
1:10,000 dilution of anti-phospho-tau antibody (Roche Products,
Mannheim, Germany), 1:2000 dilution of anti-p35 antibody (N-20; Santa
Cruz Biotechnology), or 1:2000 dilution of anti-p35/p25 antibody (C-19;
Santa Cruz Biotechnology) in blocking buffer with 0.05% sodium azide.
The blots were washed four times for 15 min each in blocking buffer and
incubated in a 1:4000 dilution of goat anti-rabbit antibody conjugated
to horseradish peroxidase (Vector Laboratories, Burlingame, CA) in
blocking buffer for 2 hr. The blots were washed eight times for 15 min
each with PBS-Tween alone, developed with the enhanced
chemiluminescence (ECL) system of Amersham (Arlington Heights, IL), and
exposed to Hyperfilm-ECL (Amersham) for 5-60 sec. Levels of protein
immunoreactivity were quantified by either measuring the optical
density of specific bands using an image analysis system with NIH Image
software, version 1.41, or measuring the light intensity using a
Bio-Rad PhosphorImager.
Cloning cdk5 promoter. Genomic DNA from mouse tail was used
as a template in PCR to clone a portion of the 5'-promoter region of
the cdk5 gene (Ohshima et al., 1996). The accuracy of the PCR product
was confirmed by DNA sequencing. Primers were designed on the basis of
GenBank sequence information of the cdk5 gene: upstream primer, 5'-CCA
GCA GCC AGA GGG GAC TCT-3', and downstream primer, 5'-AGG TGC CTA GAG
GAA GGT TG-3'. The PCR product (1.6 kb) was cloned into the
pGL3-basic vector, which contains a luciferase reporter gene.
The plasmid was designated pGL3-cdk5-luc.
Transfection. Cells (2.5 × 105) of an inducible FosB-expressing C6
glioma cell line [described in Chen et al. (1997)] in 2 ml of DMEM
containing 10% FBS were inoculated into each well of six-well Falcon
plates. After 16 hr of incubation at 37°C in 5% CO2, the cells were transfected with 2.5 µg of
pGL3-cdk5-luc DNA or pGL3-cdk5mut-luc DNA (see below) by the use of the
lipofectamine reagent (Life Technologies, Gaithersburg, MD). Cells were
harvested 24 hr later.
Site-directed mutagenesis. Primers containing mutations in
the AP-1 site of the cdk5 promoter (mutant primer 1, 5'-GGG TGT TTG TCG
ACT CCA GCG ACC TCC TGA CA-3'; mutant primer 2, GTC GCT GGA GTC GAC AAA
CAC CCA ACC AGG TCA-3') were paired with either upstream or downstream
primers for the cdk5 promoter in PCR. The AP-1 site was replaced by use
of the restriction enzyme SalI site. The PCR products were
digested with SalI and then ligated by T4 DNA ligase. The
ligated PCR product was used as a template and amplified by PCR using
upstream and downstream cdk5 primers. The PCR product containing the
mutated cdk5 promoter fragment was cloned into the pGL3-basic vector.
The mutant plasmid was designated pGL3-cdk5mut-luc.
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RESULTS |
Analysis of gene expression profiles in
FosB-expressing mice
In previous studies, we used the tetracycline gene regulation
system (Gossen and Bujard, 1992) to develop bitransgenic mice that
support the inducible expression of FosB in specific brain regions,
including the hippocampus (Chen et al., 1998) (Fig.
1A). Expression of
FosB is tightly regulated by doxycycline, an analog of tetracycline,
in bitransgenic mice. To search for downstream targets for FosB in
the hippocampus, we analyzed RNA samples derived from this brain region
of FosB-expressing mice and of their littermates not expressing
FosB, by the use of commercially available cDNA expression arrays as
shown in Figure 1, which contain 588 genes in each array. Levels of
probe hybridization to each gene were quantified by PhosphorImager (see
Materials and Methods). A portion of the resulting arrays are shown in
Figure 1C. As would be expected, levels of hybridization to
fosB DNA itself were ~4.5-fold higher with probe derived
from the hippocampus of FosB-expressing mice than with that of
doxycycline-suppressed controls (Table 1). The magnitude of this increase
corresponds to the degree of induction of FosB observed previously
in the bitransgenic mice (see Kelz et al., 1999), which in turn is
similar to that obtained with chronic ECS treatment (Hope et al.,
1994a). This result offers some validation of the ability to detect
altered gene expression by use of the cDNA expression arrays.
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Figure 1.
Identification of cdk5 as one of the
downstream target genes for FosB in the hippocampus of inducible
bitransgenic mice using cDNA expression arrays. A,
Schematic diagram of the tetracycline expression system used for the
inducible tissue-specific expression of FosB (Chen et al., 1998).
Gene 1 encodes the tetracycline transactivator (tTA)
under the control of the neuron-specific enolase (NSE)
promoter. Gene 2 encodes FosB under the control of the
tetracycline-responsive promoter with seven tetracycline operators
(TetOp). B, Strategy for searching
downstream target genes for FosB in the hippocampus of inducible
FosB-expressing bitransgenic mice. Total RNA was isolated from five
bitransgenic mice, either expressing or not expressing FosB, and
pooled. Poly(A+) RNA was isolated from the pooled
total RNA and used as a template for the synthesis of a
32P-labeled cDNA probe. The cDNA probes were hybridized to
the arrays, and the arrays were analyzed by the PhosphorImager.
C, Gene expression profiles of the hippocampus of the
bitransgenic mice, either expressing or not expressing FosB, from a
portion of the resulting cDNA expression arrays. Positions of the
FosB and cdk5 genes are indicated by arrows. The
results are representative of three independent determinations.
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By comparing the gene expression profile from bitransgenic mice
expressing FosB with the profile from mice not expressing FosB in
three independent trials, we identified genes that are consistently
upregulated or downregulated >50% after FosB expression. Of the
430 genes detected on the resulting arrays, 20 genes were reliably
upregulated by at least 50%, whereas 14 genes were reliably downregulated by at least 50%. These genes, which are listed in Table
1, encode for a wide variety of proteins, including neurotransmitter receptors and transporters and intracellular signaling proteins. Because the sensitivity of these cDNA expression arrays is much lower
than that of Northern blotting and the signals of many mRNAs detected
by the arrays were close to background, the percentage of regulation
was considered a semiquantitative measure of gene regulation.
Cdk5 is one of the downstream target genes for FosB
One of the genes identified on the DNA arrays as upregulated by
FosB is that for cdk5 (Fig. 1C), which was increased by
~61% in FosB-expressing mice (Table 1). As a first step to
confirm that this upregulation is not a false-positive result, levels of cdk5 immunoreactivity were measured by Western blotting in the
hippocampus of an independent group of FosB-expressing mice and
their doxycycline-suppressed littermates. As shown in Figure 2, levels of cdk5 were increased by close
to 50% after FosB expression.
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Figure 2.
Upregulation of cdk5 immunoreactivity in the
hippocampus of inducible bitransgenic mice after FosB expression.
A, A representative immunoblot shows cdk5 levels in the
hippocampus of bitransgenic mice expressing (+) or not expressing ( )
FosB. B, Levels of cdk5 immunoreactivity are given as
arbitrary OD units and are expressed as the mean ± SEM
(n = 5 animals in each treatment group).
*p < 0.05 by Student's t
test.
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To test whether FosB regulation of cdk5 might be a direct effect of
the transcription factor on the cdk5 gene, we cloned a 1.6 kb fragment
of the 5'-promoter of the cdk5 gene by PCR and placed it into a
reporter vector (pGL3-basic), which contains a luciferase reporter
gene. This fragment of the cdk5 promoter, as published previously
(Ohshima et al., 1996), contains several regulatory elements including
individual AP-1, AP-2, CRE, NF-IL6, and SP1 sites (Fig.
3A). The cdk5-luciferase
construct was analyzed in a stable C6 glioma cell line in which FosB
expression is under the control of the tetracycline system (Chen et
al., 1997). The cdk5 promoter exhibited strong activity in this cell
line in the absence of FosB, with 20-fold higher levels of
luciferase seen as compared with the pGL3-basic control plasmid.
Activity of the cdk5 promoter was increased by ~75% when FosB was
induced by the removal of tetracycline (Fig. 3B). To test
whether the AP-1 site in the promoter region is responsible for this
regulation, we investigated the ability of FosB to regulate a
modified cdk5 promoter in which this site was altered by site-directed
mutagenesis (Fig. 3A). As shown in Figure 3B,
mutation of the AP-1 site completely abolished upregulation of cdk5
promoter activity by FosB.
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Figure 3.
Induction of cdk5 promoter activity by FosB.
A, Schematic structure of a fragment of the 5'-promoter
of the cdk5 gene is shown. Several putative response elements within
the promoter region are indicated. The AP-1 site framed
by a rectangular box and its adjacent sequences are
shown. The AP-1 sequence in a mutated promoter
(underlined sequence) is also shown. B,
Luciferase activity was measured in a C6 glioma cell line that supports
the inducible expression of FosB (Chen et al., 1997) transfected
with the wild-type (cdk5-luc) or mutated (mutcdk5-luc) cdk5 promoter in
pGL3-basic. Data are expressed as the mean percent change in promoter
activity in the presence of FosB compared with that in the absence
of FosB (± SEM; n = 3). The results are
representative of two independent replications. *p < 0.05 by Student's t test.
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To confirm further the role of the AP-1 element in the regulation of
cdk5 promoter activity by FosB, we performed gel shift assays using
an AP-1 oligonucleotide derived from the cdk5 promoter (Fig.
4A). The results showed
robust induction of AP-1-binding activity in the hippocampus after
FosB expression (Fig. 4B). This activity was
caused by FosB, because it was disrupted by including an
anti-FosB antibody in the assay mixture (data not shown).
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Figure 4.
Upregulation of cdk5 AP-1-binding activity in the
hippocampus of inducible bitransgenic mice after FosB expression.
A, The sequence of the cdk5 AP-1 oligonucleotide used as
the probe is shown. The 32P-labeled nucleotides are
indicated by dots. B, A
representative autoradiogram shows the dramatic induction of cdk5
AP-1-binding activity after FosB expression. The results are
representative of three independent replications.
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Upregulation of cdk5 in the hippocampus by chronic
ECS treatment
Because chronic ECS treatment induces high level of FosB in the
hippocampus, it is hypothesized that chronic ECS should also upregulate
cdk5 in this brain region. To test this hypothesis, we analyzed levels
of cdk5 expression in the hippocampus of Sprague Dawley rats treated
chronically with ECS. It was found that chronic ECS treatment increased
levels of cdk5 immunoreactivity in the hippocampus by ~50% (Fig.
5A). Chronic ECS treatment was
also found to increase levels of AP-1 binding, using the AP-1
element in the cdk5 promoter, in the hippocampus by more than twofold (Fig. 5B).
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Figure 5.
Upregulation of cdk5 immunoreactivity in rat
hippocampus by chronic ECS treatment. A,
Top, A representative immunoblot shows cdk5 levels in
the hippocampus after sham or ECS treatment. Bottom,
Levels of cdk5 immunoreactivity are given as arbitrary OD units and are
expressed as the mean ± SEM (n = 8 animals in
each treatment group). B, Top, A
representative autoradiogram shows cdk5 AP-1-binding activity after
sham or ECS treatment. Bottom, Levels of cdk5
AP-1-binding activity are given as arbitrary OD units and are expressed
as the mean ± SEM (n = 8 animals in each
treatment group). *p < 0.05 by Student's
t test.
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Evidence of increased cdk5 catalytic activity after chronic
ECS treatment
Several substrates for cdk5 have been identified in brain.
Prominent among these is the microtubule-associated protein tau (Patrick et al., 1999). To test whether upregulation of cdk5
immunoreactivity is associated with an increase in its catalytic
activity, we analyzed levels of phosphorylated tau in the hippocampus
of rats treated chronically with ECS. Five phosphorylated tau isoforms
were detected by Western blotting. Each of these isoforms was
upregulated by chronic ECS treatment (Fig.
6).
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Figure 6.
Upregulation of tau phosphorylation in rat
hippocampus by chronic ECS treatment. A, A
representative immunoblot showing levels of phospho-tau proteins after
sham or ECS treatment. B-F, Levels of phospho-tau, in
arbitrary OD units, for each tau isoform ± SEM
(n = 3). *p < 0.05 by
Student's t test.
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The catalytic activity of cdk5 depends on its cofactor, termed p35,
that must be cleaved into a 25 kDa product, termed p25, to activate the
kinase (Patrick et al., 1999). To determine whether upregulation of
cdk5 immunoreactivity is associated with corresponding changes in the
levels of p35 or its p25 fragment, levels of these proteins were
analyzed by Western blotting in the hippocampus of rats after chronic
ECS treatment. Chronic ECS failed to alter p35 levels in this brain
region but did cause a significant (46%) increase in the levels of p25
immunoreactivity (Fig. 7).
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Figure 7.
Regulation of p35 and p25 immunoreactivity in rat
hippocampus by chronic ECS treatment. A, A
representative immunoblot shows p35 and p25 levels after sham or
chronic ECS treatment. B, Levels of p35 and p25
immunoreactivity are given as arbitrary OD units and are expressed as
the mean ± SEM (n = 8 animals in each
treatment group). *p < 0.05 by Student's
t test.
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DISCUSSION |
The results of the present study demonstrate, by use of DNA array
technology, that cdk5 is one of the downstream target genes for
FosB. Cdk5 immunoreactivity is increased in the hippocampus of mice
after the inducible expression of FosB within this brain region.
Binding at an AP-1 site present within the 5'-promoter region of the
cdk5 gene is also increased after FosB expression. FosB
expression also increases activity of the cdk5 promoter in
vitro, and this increase is abolished after mutation of the AP-1
site contained within the promoter. Furthermore, chronic ECS treatment,
which has been shown previously to induce FosB dramatically in the
hippocampus, also increases levels of cdk5 immunoreactivity, cdk5
AP-1-binding activity, and the state of phosphorylation of the
microtubule-associated protein tau, a known cdk5 substrate, in this
brain region.
The upregulation of cdk5 by chronic ECS treatment in the hippocampus is
an interesting finding, based on evolving evidence that chronic ECS
treatment may produce some of its clinically beneficial effects by
promoting the growth and sprouting of several types of hippocampal
neurons (Duman et al., 1997, 1999). Thus, chronic ECS increases the
expression of the neurotrophic factor BDNF in the rat hippocampus
(Nibuya et al., 1995). Chronic ECS also enhances the sprouting of
granule cell neurons in the hippocampal dentate gyrus (Vaidya et al.,
1999) and even increases the birth of new granule cell neurons (Madsen
et al., 2000). Conversely, stress exerts the opposite effects in rodent
models; it decreases BDNF expression, the sprouting of several neuronal
cell types, and neurogenesis in the hippocampus (Smith et al., 1995;
Sapolsky, 1996; Brown et al., 1999; Gould and Tanapat, 1999). These
effects can be prevented by previous treatment with ECS. Several
classes of chemical antidepressants can exert some, but not all, of the aforementioned effects. The relevance of these findings in animal models to psychiatric phenomena in humans is indicated by the observation of reduced hippocampal volume in patients with depression or other stress-related disorders (Sheline et al., 1996; Lupien et al.,
1998; Bremner et al., 2000).
Cdk5 is a plausible mediator of some of these effects of chronic ECS
administration, which is why, among all of the gene products identified
on the arrays as putative targets for FosB (Table 1), we focused
first on cdk5. Cdk5 belongs to a family of cyclin-dependent kinases
that are known to play an important role in the regulation of cell
growth (Lee et al., 1997; Zheng et al., 1998). Among this kinase
family, cdk5 is unique with respect to its enrichment in nervous tissue
and, in particular, in the fully differentiated adult brain (Hellmich
et al., 1992). Several neural proteins have been shown to be
phosphorylated by cdk5 in recent years (Julien and Mushynski, 1998;
Bibb et al., 1999; Ahlijanian et al., 2000). Prominent among these
substrates are several proteins important for neuronal structure,
including tau and neurofilament proteins (Patrick et al., 1999).
Moreover, a dominant-negative mutant of ckd5 inhibits neurite outgrowth
in primary neuronal cultures (Nikolic et al., 1996), and cdk5 knock-out
mice show abnormal development of the hippocampus and cerebral cortex
(Gilmore et al., 1998). Cdk5 also has been shown to enhance axonal
growth in cultured neurons (Paglini et al., 1998). Although further
work is needed to link causally ECS-induced upregulation of cdk5 to
enhanced sprouting and growth of hippocampal neurons, the present
results identify cdk5 as one candidate molecule that may mediate some of the long-term adaptive changes in the hippocampus induced by chronic
ECS treatment.
The enzymatic activity of cdk5 in postmitotic neurons depends on a
neuron-specific activator, p35 (Patrick et al., 1999). This effect of
p35 requires its proteolytic cleavage into p25, which directly
activates the enzyme. As a result, upregulation of cdk5
immunoreactivity in the hippocampus would be expected to result in
increased cdk5 catalytic activity only if the amount of p35 (or p25) in
this tissue is not limiting. The result that chronic ECS causes
increased phosphorylation of tau is consistent with an increase in cdk5
catalytic activity. Further evidence of this interpretation is our
finding that chronic ECS treatment also increases levels of p25
immunoreactivity in the hippocampus. The mechanism underlying this
upregulation of p25, which occurs in the absence of a detectable change
in the levels of p35, is unknown.
A novel aspect of this study is its application of DNA arrays to the
analysis of mice that support the inducible and brain region-specific
expression of a transcription factor, in this case FosB. Cdk5 is one
of 34 genes that were consistently upregulated or downregulated in the
hippocampus after expression of FosB. A major limitation of DNA
array technology, as well as of several other methodologies used to
analyze differential gene expression (such as differential display and
subtraction hybridization), is the large number of false-positive
results obtained. Although cdk5 proved to be a bona fide target for
FosB, it remains to be seen whether the other genes identified as
putative FosB targets (Table 1) are also true targets. Another
limitation with DNA array technology, at least with the filter-based
arrays used in the present study, is the relatively low sensitivity of
detection. It is quite likely that additional targets for FosB can
be found by the use of more sensitive detection methods. Our results
illustrate still another limitation of the use of DNA arrays, namely,
that after putative genes are identified by the use of arrays, one is
faced with studying the role of any individual gene by more conventional (and labor-intensive) methods. Thus, although we identified 34 putative targets for FosB, we are still obligated to
characterize the precise function of each gene one at a time.
Nevertheless, the results of the present study demonstrate the
potential power of DNA array analyses in identifying novel targets for
FosB in the brain. On the basis of available models of FosB
action, we would not otherwise have thought of cdk5 as a potential
mediator of this transcription factor. Cdk5 has been implicated
previously in the hyperphosphorylation of tau seen in certain
neurodegenerative disorders (see Baumann et al., 1993; Alvarez
et al., 1999; Patrick et al., 1999). On the basis of our findings with
DNA arrays, our results further implicate cdk5 in the neural plasticity
that accompanies the treatment of depression with chronic ECS. Such
observations could help explain the pathophysiology of depression as
well as provide new leads to the development of more effective
antidepressant treatments.
| |
FOOTNOTES |
Received July 7, 2000; revised Aug. 18, 2000; accepted Aug. 24, 2000.
This work was supported by grants from the National Institute of Mental
Health and the National Institute on Drug Abuse.
Correspondence should be addressed to Dr. Eric J. Nestler, Department
of Psychiatry, The University of Texas Southwestern Medical Center,
5323 Harry Hines Boulevard, Dallas, TX 75390-9070. E-mail:
eric.nestler{at}utsouthwestern.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
